Targeting X-linked Inhibitor of Apoptosis Protein to Overcome Rituximab/Chemotherapy Resistance in B-cell Lymphomas
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ABSTRACTTargeting X-linked Inhibitor of Apoptosis Protein (XIAP) to Overcome Rituximab/ Chemotherapy Resistance in B-Cell Lymphomas.The addition of Rituximab to front-line therapy has improved clinical outcomes in diffuse large B-cell lymphoma (DLBCL), but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for Rituximab associated chemotherapy cross-resistance our group developed and characterized several Rituximab resistant cell lines (RRCL). We previously demonstrated using SiRNA interference, that X-linked inhibitor of apoptosis (XIAP) is critical for chemotherapy sensitivity and survival in RRCL. Using MX69, a dual inhibitor of MDM2and XIAP that indirectly downregulates XIAP, in pre-clinical testing we downregulated XIAP to evaluate whether this strategy can help overcome rituximab resistance. MX69 affects XIAP levels by its effects on the ubiquitination and degradation of endogenous MDM-2, resulting in decreased XIAP translation which results in activation of caspase 3, 7 and 9, and cleavage of PARP enhancing apoptosis of cancer cells. In our current work, we pharmacologically inhibited XIAP in lymphoma pre-clinical models using MX69.In vitro, a panel of Burkitt's Lymphoma (BL, including RRCL), germinal center B-cell (GCB)-DLBCL (including RRCL), activated B-cell (ABC)-DLBCL, Mantle cell Lymphoma (MCL) and Pre-B cell Leukemia cell lines were exposed to MX69 as a single agent (0-80uM) over 24, 48, 72 hrs. Half maximal inhibitory concentrations(IC50)were calculated for each cell line, to define the single agent activity of MX69 as an antilymphoma agent. Western blotting studies confirmed the mechanism of action for the compound with downregulation of Mdm2, XIAP and changes in P53 and PARP, following in vitro MX69 exposure (at IC50 doses) for 24 hrs. Induction of apoptosis was observed by Annexin V/propidium iodine staining. Subsequently, in order to test the potential for MX69 to enhance chemosensitivity, cell lines were exposed to MX69 (0-80 uM), in combination with doxorubicin (0-1uM), cytarabine (0-50uM), vincristine (0-10nM), etoposide (0-50uM), carboplatin (0-20uM), ixazomib (0-1.5uM), ibrutinib (0-20uM) and venetoclax (0-10uM) for 48 hours. Combination treatments revealed synergy between MX69 and the chemotherapeutic agents venetoclax, cytarabine and doxorubicin. Our data suggest the anti-tumor activity of MX69 against a wide variety of B-cell lymphoma cell lines (including BL, DLBCL, MCL or RRCL) resulted in. We hypothesize that the anti-tumor effect of MX69 is due to downregulated XIAP levels. These findings support the data from our prior SiRNA XIAP knockdown experiments. Strong synergy was observed when combining XIAPsiRNAs with chemotherapeutic agents and small molecules inhibitors (such as venetoclax, cytarabine and doxorubicin). Ex vivo experiments using primary tumor cells isolated from lymphoma patients and lymphoma mouse models are planned. Targeting XIAP can be an attractive therapeutic strategy in patients with Rituximab-sensitive or -resistant B-cell lymphoma.